Gene therapy is an approach to treat diseases either by modifying the expression of one or more genes of an individual, or by correcting abnormal genes. By administration of DNA rather than a drug, many different diseases are currently being investigated as candidates for gene therapy. These include genetic disorders, e.g. cystic fibrosis, cardiovascular disease, various forms of cancer, as well as infectious diseases such as AIDS. When transferring genes to cells ex vivo or in vivo, a selection is often required, if the gene modified cells do not have a selective advantage over unmodified cells. It could also be desirable to ensure sufficient multiplication of the cells having received the new gene before transferring them to the patient.
The practical use of gene therapy is still limited due to various reasons, one of them being low gene transfer efficiency and the requirement for extended in vitro cell culture selection to obtain enriched or pure populations of gene modified cells. Different marker genes for selection of gene-modified cells have hitherto been used. These fall into two categories: cell surface markers and metabolic selection markers.
Cells modified by cell surface marker genes can be selected by fluorescence-activated cell sorting (FACS) or by immunomagnetic techniques. Cell surface markers usually allow fast selection procedures, but there is a risk of false-positive selection if the selection is performed too early following the transduction, due to transfer of the marker protein in the retroviral envelope to the target cell plasma membrane. Further, FACS is an open system leading to difficulties in maintaining sterility. In addition, sorting large amounts of cells takes considerable time. A general drawback of immunomagnetic sorting is the low recovery of gene-modified cells. Only cells with high transgene expression are efficiently sorted. Low recovery requires larger volumes of starting materials and is economically unfavourable.
Metabolic selection markers allow for efficient background-free selection of the gene-modified cells, but the duration of selection is usually long, typically lasting about one week to ten days. The selection substance may also be directly DNA damaging.
Further, there is a great risk of non-human genes or gene fragments to trigger an immune response against gene modified cells. Thus, human cells modified with these genes would be eliminated by the immune system. Immunogenicity is usually not a problem with cell surface markers since these are generally human. In contrast, metabolic selection markers are often of non-mammalian origin, documented to cause immunogenicity problems.
Another problem with current selection markers is their putative influence on normal cell functions. This poses a risk of causing cell alterations which might contribute to cell transformation.
Na+, K+-ATPase is a housekeeping enzyme present in all mammalian cells. It is an integral membrane protein that establishes the electrochemical gradient across the plasma membrane by transporting sodium ions out of the cell and potassium ions into the cell (in the ratio 3:2), utilising ATP hydrolysis as an energy source. Variations in the activity of the Na+, K+-ATPase have effects on a number of critical cell functions. The minimal functional enzyme active in the membrane is a dimer consisting of an α-subunit and a β-subunit. Four isoforms (α1, α2, α3, and α4) of the Na+, K+-ATPase α-subunit gene family have been cloned in man and rat, the α1 being the most resistant isoform to cardiac glycosides.
Cardiac glycosides are naturally occurring compounds found to have an effect on the contractive power and rhythm of the mammal heart. Examples include digoxin and digitoxin, from woolly foxglove (Digitalis lanata) and from common foxglove (Digitalis purpurea); proscillaridine A from sea squill, (Urginea (Scilla) maritime), ouabain (G-strophantine) from Strophantus gratus; convallatoxin from mayflower (Lily-of-the-valley, Convallaria majalis); and palytoxin from the coral Palythoa toxica. All cardiac glycosides seem to comprise a steroidal part, coupled to one or more glucose like molecules.
Ouabain, one member of the above group of drugs, has been shown to bind to the α-subunit and inhibits the ATPase and ion-transport activity of the enzyme. In this description, ouabain is used as one example of cardiac glycosides.
As mentioned above, current selection methods have several shortcomings negatively influencing the cell selection. Accordingly, there is a need for a rapid selection marker for use in in vitro applications as well as in human gene therapy with high efficiency and minimal immunogenicity. Such a marker would also have wide application in methods for studying the toxicology of drugs, studying the mechanisms of toxicity, screening for new drugs in vitro, etc.
One objective of the present invention is to make available such a marker, as well as methods of its production and use. Further objectives, the solutions offered by the invention, as well as their advantages will be evident to a skilled person upon study of the following description and examples.